Tick-borne encephalitis (TBE) is a neurological disease caused by Flaviviruses of the tick-borne encephalitis group and causes death in up to 60% of the individuals developing clinical symptoms. Survivors often show severe long-term neurological sequelae. In addition to the classical viruses, the TBE complex of viruses also contains members that cause hemorrhagic fevers sometimes in combination with neurological symptoms. The infection is naturally transmitted via vector ticks in endemic areas. However, the highly infective viruses can also be transmitted via food or in aerosolized form. Therefore, development of a TBE virus vaccine is a NIH high-priority biodefense project (and as such listed in the NIAID Biodefense research agenda for category B and C pathogens. Currently available commercial vaccines based on inactivated whole virus are not registered in the U.S., show considerable vaccination side-effects and are labeled for use only against the less virulent Central European subtype. In the previous phase I SBIR project recombinant TBE subunit proteins were successfully produced in insect cells. The recombinant proteins showed very potent immunogenicity in mice when used with modern adjuvants. The leading formulations showed good efficacy in the mouse challenge models of Western and Far Eastern subtype TBE viruses. In addition, a preliminary study demonstrated that the leading vaccine candidate also confers complete protection against the more distantly related Omsk Hemorrhagic Fever virus. During the phase II project, the antigen manufacturing will be advanced and the necessary quality control steps implemented to progress with the scale-up of production. This will include safety testing of well-defined antigen in a rat toxicology study. Biological and physical assays to evaluate the response to the vaccines will be further developed and standardized. Those assays will be used to document the effect of antigen dosage in the leading vaccine formulation and evaluate the need for a third vaccination to achieve complete protection (in different mouse models). Another study will compare the potency of the recombinant TBE antigen with that of conventionally produced inactivated TBE virus. After refining the vaccine formulation, protective efficacy in mice against the more distant members of the TBE complex will be evaluated. Based on mouse studies the leading candidate will be tested in non-human primates to demonstrate safety and immunogenicity. Efficacy in primates will be demonstrated indirectly using well accepted passive protection studies in mice. The planned studies should complete the pre-clinical efficacy requirements and would therefore be an important step towards the clinical development of a novel TBE vaccine. A safe and efficacious vaccine based on recombinant subunit proteins would provide useful in protecting U.S. citizens from TBEV infection without the need of large-scale culture of highly infectious virus. PUBLIC HEALTH RELEVANCE - PROJECT NARRATIVE: Tick-borne encephalitis (TBE) viruses cause severe neurological disease or hemorrhagic fevers and survivors often show severe long-term neurological sequelae. Currently available commercial vaccines based on inactivated whole virus are not registered in the U.S., show considerable vaccination side-effects and are labeled for use only against the less virulent Central European subtype. This project is aimed to develop a safe and efficacious vaccine formulation based on a recombinant subunit protein which requires only two doses to provide broad protection against all members of the TBE complex to protect U.S. citizens from TBEV infection in endemic areas around the world.